For various reasons, it is highly desirable to be able to qualitatively and/or quantitatively measure human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) in a convenient, reliable manner. For example, it is known that the concentration of hLH in female biological fluids increases dramatically just prior to ovulation. A convenient, qualitative detector of the hLH surge for "at home" use could pinpoint the time of ovulation for women with fertility problems and/or aid in fertility regulation. Also, a quantitative hLH assay, which could be used by the physician in the office would be useful in the medical evaluation and observation of various conditions associated with hLH presence and/or concentration variation.
Similarly, significant presence of hCG in the female biological fluids has been accepted as one of the most reliable tests confirming pregnancy. hCG is secreted by the developing blastocyst and can be detected in pregnant women as early as 7 to 9 days after fertilization. Again, a qualitative "at home" pregnancy test capable of providing a reliable result within the first two weeks of pregnancy would be extremely useful. Also, a convenient assay procedure usable by the practicing physician would prove of benefit in the diagnosis and evaluation of various abnormal conditions characterized by hCG levels, such as in the diagnosis of ectopic pregnancy and spontaneous abortion, and in the follow-up of patients with trophoblastic disease and infertility problems.
One aspect of the present application is concerned with improved solid phase assay systems and methods for measuring hLH and/or hCG, which do not require radioactive substances. In one embodiment of the present invention, an enzymatic marker is employed. In another embodiment, a direct dye marker is used to eliminate the step of reacting enzyme with its substrate for measurement. Another aspect of the present application is concerned with materials and reagents to be used in the improved assay systems and methods.
Van Weeman et al, "Immunoassay Using Antigen-Enzyme Conjugate", FEBS Letters, 15:232 (1971), conjugated hCG to horseradish peroxidase through glutaraldehyde and then used the conjugate for enzyme-immunoassay of hCG. A solid phase assay procedure is disclosed by Van Weeman et al where hCG antibody is attached to a cellulosic support (reprecipitated and diazotized m-aminobenzyloxymethyl cellulose or microcrystalline cellulose activated by CnBr.) Also, see U.S. Pat. No. 3,654,090 using the Van Weeman conjugate in similar assay procedures. Another enzyme used in the art in place of horseradish peroxidase is alkaline phosphatase (ALP). For example, see Engvall et al, "Enzyme-linked Immunoabsorbent Assay (ELISA), Quantitative Assay of Immunoglobulin G", Immunochemistry, 8:871 (1971). Kawaoi et al, "An Improved Method of Conjugation of Peroxidase with Protein". Fed. Proc., 32 Abstract 840 (1973) also disclose a method for forming an enzyme-hormone complex.
Saxena et al, "Development of a Solid-Phase Centrifugation-free Enzyme Assay for LH for Ovulation Detection", Psychoneuroendocrinology in Reproduction, Zichella et al, editors, Elsevier/North Holland (1979), p. 277, describe both an enzymeimmunoassay and an enzymereceptorassay for hLH using antibody-coupled glass beads or receptor-coupled glass beads, respectively (glutaraldehyde activated aminopropyl glass beads) and an hLH-alkaline phosphatase conjugate (4-azidobenzoyl derivative of hLH). The receptor used by Saxena et al was of relatively low yield and suffered from poor stability.
Carlsson et al, "Protein-Thiolation and Reversible Protein-Protein Conjugation", Biochem. J., 173:723 (1978), disclose that N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) can be employed as a heterobifunctional reagent in forming protein-protein conjugates through a disulfide link. As one example, Carlsson et al form a horseradish peroxidase-rabbit anti- (human transferrin) antibody conjugate. In general, 2-pyridyl disulfide structures are introduced into both the peroxidase and the antibodies by their reaction with SPDP. Although either of the 2-pyridyldisulfide moieties can be converted into the corresponding thiol derivative by specific reduction with dithiothreitol at pH 4.5, Carlsson et al formed the thiolated antibodies and reacted them with the peroxidase 2-pyridyl disulfide derivative by thiol disulfide exchange to produce the peroxidase enzyme-antibody conjugate.
It is also known that hLH and hCG consist of two noncovalently linked polypeptide chains classified as .alpha. and .beta. subunits. The .alpha. subunits of these glycoprotein hormones, as well as the .alpha. subunits of other glycoprotein hormones, have almost identical amino acid sequences and are practically indistinguishable immunochemically. In contrast, the .beta. subunits of each have their own distinctive amino acid sequences which differentiate them from the .beta. subunits of the other glycoproteins. Methods are known to purify antibody, such as hCG .beta. antiserum, to reduce cross-reactivity with hCG .alpha. subunits or with similar hLH subunits. For example, see Jibiki et al, "A Receptor-Immunoassay for the Determination of the Specificity of Anti-hCG-.beta. Sera", ACTA Endocrinologica, 87, 838 (1978), where hCG-.beta. antisera is purified by immunoabsorption with hLH and hCG-.alpha..
Saito et al, "Use of Receptors in the Preparation of LH-Free Serum", J. Clin. Endocrinol. Metab. 43:1186 (1976), used purified gonadotropin receptor to bind serum hLH to the exclusion of other serum proteins. This general technique provides a purer serum, depending upon the specificity of the receptor.
Glass beads have been used as the solid phase in the radioimmunoassay of hormones such as hLH and hCG. See Post et al, "A Rapid, Centrifugation-Free Radioimmunoassay Specific for Human Chorionic Gonadotropin Using Glass Beads as Solid Phase", J. Clin. Endoclinol. Metab., 50:169 (1980), where solid glass beads of 6 mm diameter were sandblasted and then heated in the presence of .gamma.-aminopropyltriethoxysilane to generate reactive alkylamino groups on the glass surface. The amino groups are activated with glutaraldehyde and subsequently hCG-.beta. antibody is covalently coupled thereto.
Finally, a key reagent in an hLH or hCG receptor assay is the receptor itself. Heretofore, large quantities of the common hLH and hCG receptor of the corpora lutea have not been obtained in sufficiently pure form for use in enzyme receptor assays of good reliability and accuracy. See Khan et al, "Use of Purified Gonadotropin Receptor in the Development of an Enzyme Receptorassay (ERA) for Luteinizing Hormone (LH) and Human Chorionic Gonadotropin (hCG)", Enzyme Labelled Immunoassay of Hormones and Drugs, S. B. Pal, editor, de Gruyler & Co. (1978).
A number of the steps used in the receptor purification system of the present invention, the present invention resulting in a good yield of a receptor having better stability and higher purity, were disclosed by the inventors in the symposium paper "Current Status of the Purification and Characterization of LH-HCG Receptors," Functional Correlates of Hormone Receptors in Reproduction, Mahesh et al, editors, page 397, Elsevier North Holland (1980), presented Oct. 15, 1980.
U.S. Pat. No. 4,016,250 and as a divisional thereof, U.S. Pat. No. 4,094,963, both to Saxena, disclose receptorassay methods for determining hCG and/or hLH in a biological fluid wherein binding of said hCG and hLH in the sample is with a plasma membrane extract from the corpus luteum of a species possessing the common receptor for hCG and hLH. The additional purification steps disclosed herein, as compared to the Saxena patents, result in the preparation of a receptor material of significantly higher purity and binding capacity.